Fungus wherein the areA gene has been modified and an areA gene from Aspergillus oryzae

ABSTRACT

A modified Aspergillus oryzae cell having an area gene which does not express a functional AreA activator, DNA sequences encoding the area gene, and method for using the cell of the invention for production of a heterologous polypeptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. 371 national application ofPCT/DK95/00254 filed Jun. 19, 1995, which is incorporated herein byreference.

FIELD OF THE INVENTION

The present invention relates to fungi, which do not produce proteases.The fungi of the invention are useful as hosts for the production ofproteins susceptible to proteolytic degradation by the proteases usuallyproduced, and the invention consequently encompasses processes for theproduction of proteins of interest in high yields by using the fungi ofthe invention. The invention also comprises methods for producing suchfungi and DNA constructs to be used in these methods.

BACKGROUND OF THE INVENTION

Fungi, and especially filamentous fungi, are widely used commerciallybecause of their ability to secrete remarkably high levels of proteins

Among the filamentous fungi species belonging to the genus Aspergillushave a long history of commercial use for the production of endogenousand lately also heterologous proteins.

One disadvantage with most microorganisms used for the production ofproteins is the inherent production of proteases which may subject aprotein product of interest to degradation due to proteolysis.

Various ways of avoiding this have been envisaged. Among other solutionsit has been suggested to delete or disrupt the genes encoding thevarious proteases. Unfortunately the fungi produce a high number ofproteases making such a solution more or less unrealistic.

A need is therefore persisting for strains of filamentous fungiexhibiting no or very low levels of protease production.

For a number of years it has been known that the regulatory gene areAwhich mediates nitrogen metabolite repression in A. nidulans influencesthe production of extracellular proteases (Arst & Cove, molec. gen.Genet. 126, (1973) 111-141).

The areA gene from A. nidulans has been cloned (Caddick et al., EMBOJournal 5, (1986) 1087-1090) and various modifications made to it toevaluate functions of different regions in the activator protein encodedby this gene (Stankovitch et al. Mol. Microbiol. 7, (1993) 81-87).Furthermore the gene coding the corresponding function in A. fumigatusapparently has been cloned recently (Hensel et al. 2nd EuropeanConference on Fungal Genetics, Apr. 28 to May 1, 1994, Book ofAbstracts, E11).

From the literature a single use is also known of a strain of A.nidulans of genotype argB areA1 as a host for the production of t-PA(Upshall et al. Biotechnology 5, (1987) 1301-1304). In this example onlythe argB genotype is used as a selection marker through its arginineprototrophy, while the area genotype is simply a coincidence.

The present invention has as an object the alleviation of the need forprotease free filamentous fungi.

SUMMARY OF THE INVENTION

The present invention consequently relates to fungi, wherein the areAgene by recombinant DNA technology has been modified such that it cannotbe expressed in a way providing for a functional AreA activator.

The invention furthermore relates to methods for producing such fungi,obtained by deletion of the areA gene.

This may be obtained through a method comprising

i) cloning of the areA gene from a fungus of interest,

ii) producing a DNA construct comprising the areA gene wherein aninternal part has been substituted, deleted, or extra DNA has beeninserted,

iii) transforming said fungus with the construct, and

iv) selecting transformants which are areA.

The information obtained from the above mentioned cloning of the areAgene may also be used in connection with the well-known anti-sensetechnology, to construct an expression plasmid giving rise to synthesisof a RNA molecule complementary to the mRNA transcribed from the areAgene, and to transform the fungus of interest therewith.

The invention furthermore relates to DNA constructs intended for use inthe above mentioned methods.

Furthermore the invention relates to methods of producing a desiredprotein or gene product, especially secreted proteins, whereby a fungalhost modified and optionally transformed with a DNA construct comprisingat least a DNA sequence coding for the protein or gene product ofinterest, is cultivated in a suitable growth medium at appropriateconditions and the desired gene product is recovered and purified.

When working with the invention it was surprisingly found that the fungiof the invention produces such secreted proteins in a much improvedyield.

It was also surprisingly found that the only nitrogen source capable ofproviding good growth of the A. oryzae areA strains was glutamine.

Lastly the invention relates to protein products produced by the abovemethods.

BRIEF DESCRIPTION OF THE DRAWING

The invention is described in further detail in the following parts ofthe specification with reference to the Examples and the drawing,wherein

FIG. 1 shows the steps involved in the construction of HowB101,

FIG. 2 shows the steps involved in the construction of pSK5 and pSK9,

FIGS. 3a and 3b show the steps involved in the construction of pToC266,

FIG. 4 shows the steps involved in the construction of pMT1606, and

FIG. 5 shows the steps involved in the construction of pToC56.

DEFINITIONS

In the present specification the following definitions are used

The expression areA.increment. means a strain in which the areA gene isdeleted.

The expression areA means a strain which does not produce a functionalAreA activator. The term "loss of function" is also often used for this.

The expression "anti-sense technology" describes methods such asdisclosed in U.S. Pat. No. 5,190,931.

DETAILED DESCRIPTION OF THE INVENTION

As indicated the present invention relates in its first aspect to fungi,wherein the areA gene by recombinant DNA technology has been modifiedsuch that it cannot be expressed in a way providing for a functionalAreA activator.

This object may specifically be obtained by deletion or disruption ofthe areA gene.

The cloning of the areA gene is described in the Examples.

AreA homologs from other fungi could be cloned either by crosshybridization with one of the already known genes or by complementationof areA mutants; e.g. A. nidulans areA-18 or the A. oxyzae areA deletedstrain described in this application.

Methods for deleting or disrupting a gene are specifically described inWO 90/00192 (Genencor).

Methods for substituting DNA in a gene are also generally known, and canbe accomplished by substituting one or more continuous parts of thegene, but it may also be obtained by site directed mutagenesisgenerating a DNA sequence encoding a AreA activator variant that is notfunctional.

Another method by which such an object may be obtained is by usinganti-sense technology.

The anti-sense technology and how to employ it is described in detail inthe aforementioned U.S. Patent No. 5,190,931 (University of New York).

A further method of obtaining said inactivation is by inserting extraDNA internally in the areA gene, thereby giving rise to the expressionof a dysfunctional activator protein.

In connection with this method information provided by the cloning canbe used to make DNA constructs that can be integrated into the areAgene, and even replace it with another gene, such as the pyrG gene.

A further method of avoiding the presence of the area activator is byinterfering with the regulation of the expression signals regulating theexpression of the areA gene itself.

According to the invention the fungus preferably belongs to a genusselected from the group comprising Aspergillus, Trichoderma, Humicola,Candida, Acremonium, Fusarium, and Penicillium

Among these genera species selected from the group comprising A. oryzae,A. niger, A. awamori, A. phoenicis, A. japonicus, A. foetidus, A.nidulans, T. reesei, T. harzianum, H. insulens, H. lanuginosa, F.graminearum, F. solani, P. chrysogenum, and others are preferred.

As indicated the invention also is meant to encompass the method forproducing the fungi of the first aspect of the invention, and whereinsaid inactivation has been obtained by deletion of the areA gene, whichmethod comprises

i) cloning of homologues of the area gene from a fungus of interest,

ii) producing a DNA construct comprising the areA gene wherein aninternal part has been substituted, deleted, or extra DNA has beeninserted,

iii) transforming said fungus with the construct, and

iv) selecting transformants which are areA.

Also included is the method for producing the fungi, wherein theinactivation has been obtained by using anti-sense technology. Such amethod comprising

i) construction of an expression plasmid which gives rise to synthesisof a RNA molecule complementary to the mRNA transcribed from the areAgene,

ii) transformation of the host fungus with said expression plasmid and asuitable marker, either on separate plasmids or on the same plasmid,

iii) selection of transformants using said marker, and

iv) screening transformants for strains exhibiting a reduction in thesynthesis of the AreA product, e.g. by analysis of the growth on variousnitrogen sources.

A further aspect of the invention is meant to comprise DNA constructsfor use in the above mentioned methods.

In respect of the former method said DNA constructs may comprise theareA gene wherein an internal part has been substituted, deleted, orextra DNA has been inserted.

The DNA construct may furthermore also comprise DNA sequences encoding aprotein product of interest, such as those mentioned later.

In respect of the latter anti-sense method the DNA construct maycomprise an inverted DNA sequence of the areA gene connected to afunctional promoter, whereby the mRNA is at least partiallycomplementary to mRNA produced from the area gene.

A further aspect of the invention relates to a process for theproduction of a desired gene product, preferably a secreted geneproduct, whereby a fungus according to the invention is cultivated in asuitable growth medium at appropriate conditions and the desired geneproduct is recovered and purified.

In the case of a gene product expressed by a heterologous gene the DNAsequence coding for the desired gene product may be a part of the DNAconstruct used for producing said fungus.

Normally, however, a separate transformation of the fungus of theinvention is performed in order to make the fungus capable of producingthe desired product.

Methods for transforming fungi are well known in the art, cf. e.g. EP 0184 438 A2 (Gist-Brocades N.V.) and EP application no. 87103806 (NovoNordisk A/S) and.

For indigenous products this is of course not necessary, but in order toincrease the production it may be an advantage to provide for multiplecopies of the gene encoding the protein of interest to be incorporatedinto the host.

The desired gene product is generally a peptide or protein, preferablyan enzyme.

Among enzymes it is preferably selected from the group comprisingproteases, such as trypsin and chymosin; lipases, cutinases, cellulases,xylanases, laccases, pectinases, etc.

Another type of desired gene product is generally a therapeuticallyactive peptide or protein.

Among the therapeutically active peptide or protein the proteinpreferably is selected from the group comprising insulin, growthhormone, glucagon, somatostatin, interferons, PDGF, factor VII, factorVIII, urokinase, t-PA, CSF, lactoferrin, TPO etc.

The invention is explained in further detail in the Examples givenbelow. These should, however, not in any way be construed as limitingthe scope of the invention as defined in the appended claims.

EXAMPLES Materials and Methods

Strains

A. oryzae, IFO4177: available from Institute for Fermentation, Osaka;17-25 Juso Hammachi 2-Chome Yodogawa-Ku, Osaka, Japan.

ToC913: The construction of this strain is described in the Examples.

Genes

areA: This gene codes for a regulatory protein controlling nitrogencatabolism.

pyrG: This gene codes for orotidine-S' -phosphate decarboxylase, anenzyme involved in the biosynthesis of uridine.

bar: This gene was originally isolated from Streptomyces hygroscopicusand codes for phosphinothricin acetyltransferase. The enzyme modifiesphosphinothricin (=glufosinate) and thereby inactivates this compoundwhich is toxic to bacteria, fungi and plants.

Plasmids

PUC118: Viera and Mesing J. Meth. Enzymol. 1987 153 3-11

pS02: The construction of this plasmid is described in the Examples.

pJers4: A 2.0 kb subclone of pS02 in pUC118. pJers4 contains afunctional A. oryzae pyrG gene.

pS05: The construction of this plasmid from pS02 is described in theExamples.

pToC56: The construction of this plasmid is described in EP applicationno. 87103806.

pToC266: The construction of this plasmid is described in the Examples.

pMT1606: The construction of this plasmid from pBP1T (B. Straubinger etal. Fungal Genetics Newsletter 39(1992):82-83) and p775 (EP applicationno. 87103806) is described in the Examples.

p777: The construction of this plasmid is described in EP applicationno. 87103806.

pHW470: The construction of this plasmid is described in the Examples.

EXAMPLE 1

Construction of an Aspergillus oryzae areA.increment. strain.

The areA.increment. strain was constructed by the following steps. TheA. oryzae pyrG gene was cloned and an A. oryzae pyrG mutant strain wasisolated. The areA gene from A. oryzae was cloned. The pyrG mutant wastransformed with a plasmid carrying the pyrG gene inserted between DNAfragments upstream and downstream from the areA gene. The coding regionfor areA was not present on the plasmid. Transformants were selected fortheir ability to grow in the absence of uridine and in the presence ofchlorate. This double selection selects both for a functional pyrG geneand for areA minus. Strains obtained by this selection procedure werefinally screened by Southern analysis to identify those in which thechromosomal areA gene was substituted by the pyrG gene.

Cloning of the A. oryzae pyrG gene.

The A. oryzae pyrG gene was cloned by cross hybridization with the A.niger pyrG gene (W. van Hartingsveldt et al., Mol. Gen. Genet 206:71-75(1987)). A lambda library of partial SauIIIA digested A. oryzae IFO4177DNA was probed at low stringency with a 1 kb DNA fragment from the A.niger pyrG gene. A 3.8 kb HindIII fragment from a positive clone wassubcloned into a pUC118 vector. The resultant plasmid, pSO2, was shownto contain the pyrG gene by complementation of an A. niger pyrG mutant.

Construction of an A. oryzae pyrG minus strain.

A pyrG deletion plasmid, pSO5, containing about 1 kb of pyrG flankingsequences on each end was constructed from the plasmid pSO2. A. oryzaeIFO4177 was transformed with this construct and transformants wereselected by resistance to 5-fluoro-orotic acid, a phenotypecharacteristic of pyrG mutants. One transformant, HowB101, was shown bySouthern analysis to have the expected deletion at the pyrG locus. Beinga pyrG mutant HowB101 requires uridine for growth. HowB101 can betransformed with the wt pyrG gene by selection for ability to growwithout uridine.

The steps involved in the construction of HowB101 are illustrated inFIG. 1.

Cloning of the areA gene.

The A. oryzae areA gene was cloned by cross hybridization to the A.nidulans areA gene (B. Kudla et al., EMBO J. 9:1355-1364 (1990)). Agenomic library of A. oryzae IFO4177 was prepared by partial digestionof chromosomal DNA with SauIIIA and cloning of the obtained DNAfragments into the vector λGEM-II (obtained from Promega). Crosshybridization of the library with the A. nidulans areA gene wasperformed in 40% formamide at 37° C. Hybridizing λ clones were isolatedand from these fragments were sub-cloned into the vector pBluescript SK+(obtained from Stratagene) giving rise to the plasmids pSK5 and pSK9illustrated in FIG. 2. The cloned gene was able to complement an A.nidulans areA mutant, proving that it is indeed the A. oryzae areAhomolog. 5643bp of the clone was sequenced, and comparison of thesequences of the A. oryzae and the A. nidulans areA genes shows thatthey are highly homologous. The sequence of the A. oryzae areA gene isshown in SEQ ID No. 1.

Construction of the areA deletion plasmid.

In order to delete the areA gene from the A. oryzae chromosome theplasmid pToC266 was constructed. pToC266 contains a 2.1 kb DNA fragmentoriginating upstream of the areA gene (isolated from pSK5) and a 1.4 kbDNA fragment originating downstream from the areA gene (isolated frompSK9). The two fragments are separated by appr. 3.2 kb in the genome,the coding region is situated in this part of the gene. The A. oryzaepyrG gene from pJers4 was inserted between the areA upstream anddownstream DNA fragments. The construction of pToC266 is illustrated inFIGS. 3a and 3b. pToC266 has a unique EcoRI site and was linearizednearized by cutting with this restriction enzyme before used intransformations.

Selection of A. oryzae areA.increment. strains.

A. oryzae HowB101 was transformed with linearized pToC266. Transformantswere selected on minimal plates (Cove Biochem. biophy. Acta (1966) 11351-56) containing 5% sodium chlorate and 0.5 mM ammonium sulfate and 1%glucose. Transformants were thus subject to a double selection, both forhaving obtained the pyrG gene by being able to grow without addition ofuridine and for chlorate resistance. Chlorate resistance is one of thephenotypes of A. nidulans areA mutants (H. N. Arst and D. J. Cove, MGG126:111-141 (1973)). Weakly growing transformants were reisolated twiceon the same type of plates. Three independent transformants namedToC913, ToC919 and ToC920 were subjected to growth test on differentnitrogen sources. They grew well on glutamine, but weakly on othernitrogen sources tested, including ammonia. Southern analysis showedthat the three strains have the lost the areA structural gene, which hadbeen replaced by the pyrG gene.

areAA strains can also be obtained by selection of transformants oflinearized pToC266 on minimal plates containing glutamine as nitrogensource. In one such experiment one out of 25 transformants was anareA.increment. strain.

EXAMPLE 2

Construction of pMT1606

A plasmid containing the bar gene from Streptomyces hygroscopius (C. J.Thompson et. al, EMBO J. 6 : 2519-2523 (1987)) inserted after the A.oryzae TAKA-amylase promoter and followed by a fragment containing thetranscriptional terminator and polyadenylation signal from the A. nigergla gene was constructed.

The plasmid, pMT1606, can be used for selection of glufosinate resistanttransformants of A. oryzae. pMT1606 was constructed by isolating the bargene from the plasmid pBP1T (B. Straubinger et. al, Fungal GeneticsNewsletter 39:82-83 (1992)) and cloning it into the fungal expressionplasmid p775 described in EP application no. 87103806. FIG. 4illustrates the construction of pMT1606.

EXAMPLE 3

Production of chymosin in ToC913 (A. oryzae IFO4177 areA.increment.)

The A. oryzae areA.increment. strain ToC913 was transformed with theplasmid pToC56 (FIG. 5), which is a fungal expression plasmid for themammalian enzyme chymosin, by co-transformation with pMT1606.Construction of the plasmid pToC56 is described in EP application no.87103806.

Transformants were selected for growth on minimal medium containing 10mM ammonium and 1 mg/ml glufosinate and screened for the presence ofpToC56 by the ability to produce chymosin. Three transformants weregrown in shake flasks in minimal medium containing maltodextrin andglutamine for 4 days at 30° C.

Two transformants of pToC56 in IFO4177 (obtained as described in EP87103806) as well as untransformed IFO4177 and ToC913 were grown alongwith the ToC913 transformants.

Samples of the fermentation broth were taken every day and applied toSDS-Page and Western blotting. The blotting membrane was incubated withchymosin specific rabbit antibody followed by goat rabbit antibodycoupled to peroxidase. Staining of the membrane showed that thesupernatants from transformants of IFO4177 contained small amounts ofchymosin or degradation products thereof on the first and second day offermentation and nothing later in fermentation.

Transformants of ToC913 contained at least ten times more full sizechymosin. The amount of chymosin in the supernatants increased for thefirst two-three days and then remained constant.

Supernatants from the third and fourth day of fermentation of IFO4177,ToC913, a transformant of pToC56 in ToC913, and a transformant inIFO4177 were applied to an isoelectric focussing gel and electrophoresiswas performed. The pH gradient was from 3.5 to 9.5. Afterelectrophoresis the gel was rinsed with a buffer at pH =7.0 containing 2mM Zn² + and overlayed with an agar containing 0.5% casein. The gel wasincubated at 45° C. untill protease activity was visible.

In samples from IFO4177 three bands with protease activity could beseen; one with an alkaline pI and two with acidic pI's.

In samples from the pToC56 transformant of IFO4177 a faint reaction fromchymosin could be seen, which partially overlapped with one of theacidic bands found in untransformed IFO4177, the protease with mostacidic pI was barely visible, while the protease with the alkaline pIwas clearly visible along with one or more band with an almost neutralpI.

In the samples from ToC913 no protease activity was detected, while thesample from the pToCS6 transformant of ToC913 showed a strong chymosinsignal. No other proteases were detected in samples from thistransformant.

EXAMPLE 4

Production of human trypsin I in ToC913 (A. oxyzae IFO4177areA.increment.)

A cDNA encoding human pancreatic trypsinogen I (TRYI) was isolated usingstandard procedures and the sequence published by M. Emi et al, Gene(1986) 41:305-310(cf. Danish patent application no. 693/95). A BamH1site (GGATCC) was introduced immediately upstream of the start codon(ATG(Met)) with the short sequence ACC between.

This BamH1 site was used to fuse the cDNA to the BamH1 linker in theTaka-amylase promoter in the fungal expression plasmid p777 described inEP application no. 87103806. The 3' end of the cDNA was fused 41 bpdownstream of the stop codon to a Nru1 site in p777. This inserts theTRYI cDNA between the A. oryzae Taka-amylase promoter and the A. nigerglucoamylase transcription terminator. The resulting plasmid was calledpHW470 (cf. Danish patent application no. 693/95).

pHW470 was transformed into ToC913 by co-transformation with the plasmidpMT1606. BASTA resistant transformants were reisolated twice throughconidiospores. 8 transformants were grown for four days at 30° C. in YPM(YPD(Sherman, F. et al (1981) Methods in Yeast Genetics. Cold SpringHarbor Laboratory. Cold Spring Harbor, N.Y.)) in which the glucose wasreplaced with 2% maltose). Supernatants were analysed for the content ofhuman trypsin by SDS-PAGE followed by Western blotting and incubationwith a rabbit antibody raised against porcine trypsin. The blottingmembrane was then incubated with goat anti rabbit antibody coupled toperoxidase and reacted with 3-amino-9-ethyl carbazole. Supernatants fromthree of the transformants contained a stained band of the expectedsize. The concentration of trypsin in the three positive supernatantswas 2-5 mg/I.

The presence of trypsin was further verified by incubation of samples ofsupernatants with L-Benzoyl-arginoyl-paranitro anilide (L-BAPNA).Samples from the three immuno positive strains cleaved the substrate,which resulted in the development of a yellow colour. Samples fromToC913 and IFO4177 did not show any activity against this substrate. Thespecific activity of human trypsin in this assay in not known, it isthus not possible to calculate the concentration of trypsin in thesupernatants from these data.

Transformants of pHW470 in the wild type strain IFO4177 were also made.More than 20 L-BAPNA positive transformants were looked at, but it wasnot possible to detect any immonoreactive bands in supernatants fromthese transformants. The detection limit was approximately 0.5 mg/l inthis assay.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 2                                           - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5643 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -     (vi) ORIGINAL SOURCE:                                                          (A) ORGANISM: Aspergillus - #oryzae                                           (B) STRAIN: IFO4177                                                  - -     (ix) FEATURE:                                                                  (A) NAME/KEY: intron                                                          (B) LOCATION: 2701..2769                                             - -     (ix) FEATURE:                                                                  (A) NAME/KEY: CDS                                                             (B) LOCATION: join(2282..2 - #700, 2770..4949)                       - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                           - - AAGCTTCGTC CTCGCATCTC GGCCGGGTGA GTAAGGTATG GTATTATTCA TG -            #AAGGGATC     60                                                                 - - TCGTTGGTTA CCGTTGTCTA TCCCTAAACA AAGGATTCAA GAGAACAACT CG -            #GAATGCTC    120                                                                 - - CCTCCGCTTA AACCCCTTGA CTCACTGATG GTGTATGTAC TATGGGTACG AC -            #GTTCGGGA    180                                                                 - - TGTGGACTAC CAACCAGAGA GTGATTAGAG AGTCCGGGTT CTCAGTCCAT GA -            #TTTTTGCA    240                                                                 - - TCTTTGAAAC AGACGATGCG GAGCGGTCAT TGGCGGAGTT TACTCCCAAA TA -            #CGGCCGAA    300                                                                 - - CGGGGTACTT TAAGTGGAAT CTCCGATTTT GGATCTAAGC TCATGAAGGA AA -            #AGTACTAC    360                                                                 - - TAATGCGTAC CTGTGCCTAA TGTTAGTGCT AGTTCGTCTG TTGCATTTTA CC -            #CGTCGGTT    420                                                                 - - AAGACGAATG GATCCGTTCA GGTTTTAAAA TAACTATCTA TGAAATATTT TA -            #GATTTCCC    480                                                                 - - GACATAGTGG TTGGGATGTC TCGATTAACA CTAGGTACAT CAGGCTCAAT TG -            #ATTTTGGT    540                                                                 - - TTTAACGAAA CATGATATAG GTCAGGGTCG TGGACCACCC TCCGCCAGGG AT -            #CAGGGGAC    600                                                                 - - GGTTACATGC GAAGGATTCT GATTATATTC ATGATTATGT CAAGCCTTTT CT -            #CTCGTGTG    660                                                                 - - AAGAGGAGCA GAGAATCCGT ACGGGTTTAA TTTAATTTAG CGCCCTGCAG CT -            #TCGAGAAC    720                                                                 - - ATCCCCAGCA ACGTTAAAAA CCACGAGCTA AAATGGGTCG CCACCGGAAG CA -            #CTCGAGTC    780                                                                 - - GAGAGATCGG TCGGCTCAGT ATTCGTAATA CCTGCGTTCC AGACGGTTTT GG -            #TCGTTGGT    840                                                                 - - TTCACTCAGG GAACTTAATT CCAGCGGGAC CCAATATAAT TTGAATGATT CA -            #TGATACAT    900                                                                 - - CCATTCGTTT GAACCGATCC TGCAAGAGTT CTGTCTGATT TGGTCAACAT AG -            #TTTTCCTC    960                                                                 - - TGGGGGAGAC TGGGGAAGAG TCAACACAAT GGTCAGGGAG AGAAGAATGA AA -            #GCTCTCGC   1020                                                                 - - AAGTGGATGA TCATGCTACG TACTGTAGGA ATAAAATTAA TTAATGCGAG GC -            #TGCAAGTA   1080                                                                 - - TCCCTGCGCC GATTTTCTCT TCTTACGGCG GGAACCAAAA AATGTGACGC TG -            #TGATTTTC   1140                                                                 - - TGGAAAAGGT AAGGATGTTT AGTTTCCCAG GATTATTACT GGTTCCGTAT GT -            #GTATGTGT   1200                                                                 - - ATGGATATCA TTCCGTATGG ATACGCCCGT TTCCTCCGCC CAGAACCAGT CC -            #GTCATCCA   1260                                                                 - - TCCTCCACTC TTTCTTCTCT TAGAGCCTTT CCACCTCTCT TCACTTTCTT TT -            #TCTTTCCC   1320                                                                 - - CCCTCCCTCT TTGCTTTCCC TCTCCCAGTA TTATTCTTAT ATTATCGGTT TG -            #ACCGTCGC   1380                                                                 - - CTCAGTATCG GCCCCCCGTG AATCACTTTT CGTTTCTCTT GTATTTTACT TT -            #CCTATCTG   1440                                                                 - - GGATTGCTCC TCGATTAGCA GCTCTACTTC ATTCGGCCAT GTGCGTCTAG AG -            #GGTCTAGC   1500                                                                 - - CCCTCTCTCT CTTTGCACTG ACTGTCAGCC ATACCATAGT ATCATCCCGG AA -            #TTAAGAAA   1560                                                                 - - AAAAAAGAAA TTATTCTACC TCCGATCTGG ACAAATTATA ACCAGGAGAA AA -            #TCAAGCGA   1620                                                                 - - AAGAGGGGCA AAGGAGGAGA CACCATTAAA ACTGGGTCTG GTTTGATTCA TG -            #ACATACAT   1680                                                                 - - TCGTCGTCTT GAATTTCAAT AGGTACGGAC TGATGCATTC CACTCGAGCC TT -            #TTTAGCTG   1740                                                                 - - CGTGTCCGTC TCCAATCGCA CTTCTTTTCT TATTTCCTTG TGGGATAAAT TG -            #ATTATTTA   1800                                                                 - - CCGTTTCGTT TTCTCTATAT TGCGGTGGTG GTGCGACCCA TCCAACTATT AT -            #TATTATAA   1860                                                                 - - TTGGAATTTG ATTTGGATTT TGATTCCTGT GACGGATCTC AGACCAAGTG CC -            #TAAACTAT   1920                                                                 - - AACTGACTTG GACCCCCTTC AGATCCTAGC TTCCCGATTC TTTTCCACCA CT -            #GCTGCATC   1980                                                                 - - CTCTTCCTGC ACGCAGCGTT CGTTTAGGGC GGGTAGACTG GAATTTATTC CT -            #TGCGCCAC   2040                                                                 - - GGACCAATCG CTCCCTCGAC GCTCTCATTC CTGCGTCGAG CTCTTTTTCC CT -            #CGACTCTC   2100                                                                 - - ATTGCTTGCT GGGCTGGTTC TTGAACCTCT TCAATCGTCC TTATCTCTTT CC -            #CCCCATCC   2160                                                                 - - GGCCTGTGAT TCCTATCTTT CCTTTTTTTC TTCCCTTTCT TGTTTGATCC CC -            #CCTCCTCC   2220                                                                 - - CCGTCTTATC GCCTACTATC GTGATCCCCG CCCTTCCCAA TAAAGAGTAG GG -            #CGTGTGAA   2280                                                                 - - C ATG TCC GGG TTA ACC CTC GGG CGA GGC CCT - #GGG GGC GTG CGA CCG           2326                                                                         Met Ser Gly Leu Thr Leu Gly Arg Gly P - #ro Gly Gly Val Arg Pro                 1              - # 5                 - # 10                 - # 15         - - ACT CAA ACC GCA ACT TTT ACC ACC CAC CAC CC - #G TCC GCC GAT GCT GAC         2374                                                                       Thr Gln Thr Ala Thr Phe Thr Thr His His Pr - #o Ser Ala Asp Ala Asp                            20 - #                 25 - #                 30              - - CGC TCC TCC AAC AAC CTC CCC CCT ACC TCC TC - #G CAG CTG TCC GAT GAC         2422                                                                       Arg Ser Ser Asn Asn Leu Pro Pro Thr Ser Se - #r Gln Leu Ser Asp Asp                        35     - #             40     - #             45                  - - TTT TCT TTC GGT TCC CCT CTG AGC CCC GCC GA - #C TCA CAG GCC CAT GAC         2470                                                                       Phe Ser Phe Gly Ser Pro Leu Ser Pro Ala As - #p Ser Gln Ala His Asp                    50         - #         55         - #         60                      - - GGC CTA CTT CAG GAC TCC CTC TTC CCT GAA TG - #G GGG TCT GGT GCG CCT         2518                                                                       Gly Leu Leu Gln Asp Ser Leu Phe Pro Glu Tr - #p Gly Ser Gly Ala Pro                65             - #     70             - #     75                          - - CGA CCC GGC ATT GAC AGT CCG GAT GAG ATG CA - #G AGG CAA GAT CCG CTA         2566                                                                       Arg Pro Gly Ile Asp Ser Pro Asp Glu Met Gl - #n Arg Gln Asp Pro Leu            80                 - # 85                 - # 90                 - # 95       - - GCG ACT CAA ATA TGG AAG CTC TAT TCT AGG AC - #C AAG GCC CAG TTG CCC         2614                                                                       Ala Thr Gln Ile Trp Lys Leu Tyr Ser Arg Th - #r Lys Ala Gln Leu Pro                           100  - #               105  - #               110              - - AAC CAG GAG CGT ATG GAA AAC CTG ACC TGG CG - #G ATG ATG GCG ATG AGT         2662                                                                       Asn Gln Glu Arg Met Glu Asn Leu Thr Trp Ar - #g Met Met Ala Met Ser                       115      - #           120      - #           125                  - - TTG AAA CGT AAG GAG CGG GAA CGT GCT CAA CA - #G TCC  AT  GTAGGTGTTC         2710                                                                       Leu Lys Arg Lys Glu Arg Glu Arg Ala Gln Gl - #n Ser  Met                              130          - #       135          - #        140                     - - TCCCTCTGTA GAGGAACGGC TGGACCCGCT CATCATTAAT TTTTTTTTTG TC -             #TGTGAAG G  2770                                                                 - - TTT CCT GCG AGA CGC GGT AGC GCT GGC CCC AG - #T GGT ATC GCT CAA        CTG     2818                                                                    Phe Pro Ala Arg Arg Gly Ser Ala Gly Pro Se - #r Gly Ile Ala Gln Leu                          145  - #               150  - #               155              - - CGC ATT TCC GAC CCG CCC GTT GCC ACC GGT AA - #C CCT CAG TCA ACC GAC         2866                                                                       Arg Ile Ser Asp Pro Pro Val Ala Thr Gly As - #n Pro Gln Ser Thr Asp                       160      - #           165      - #           170                  - - CTG ACC GCC GAC CCT ATG AAC CTC GAC GAT TT - #C ATC GTG CCC TTC GAA         2914                                                                       Leu Thr Ala Asp Pro Met Asn Leu Asp Asp Ph - #e Ile Val Pro Phe Glu                   175          - #       180          - #       185                      - - TCT CCT TCG GAC CAC CCC TCG CCC AGT GCC GT - #C AAG ATT TCC GAC TCC         2962                                                                       Ser Pro Ser Asp His Pro Ser Pro Ser Ala Va - #l Lys Ile Ser Asp Ser               190              - #   195              - #   200                          - - ACG GCG TCC GCG GCC ATT CCC ATC AAG TCC CG - #G AAA GAC CAG CTG AGA         3010                                                                       Thr Ala Ser Ala Ala Ile Pro Ile Lys Ser Ar - #g Lys Asp Gln Leu Arg           205                 2 - #10                 2 - #15                 2 -      #20                                                                              - - GAT TCT ACC CCG GTG CCG GCC TCG TTC CAC CA - #T CCG GCT CAG GAT        CAA     3058                                                                    Asp Ser Thr Pro Val Pro Ala Ser Phe His Hi - #s Pro Ala Gln Asp Gln                          225  - #               230  - #               235              - - CGG AAG AAC AGT GAA TTT GGC TAC GTC CCC CG - #T CGC GTG CGC AAG ACG         3106                                                                       Arg Lys Asn Ser Glu Phe Gly Tyr Val Pro Ar - #g Arg Val Arg Lys Thr                       240      - #           245      - #           250                  - - AGT ATC GAC GAG CGT CAA TTT TTC TCA CTG CA - #G GTG CCG ACC CGA AAG         3154                                                                       Ser Ile Asp Glu Arg Gln Phe Phe Ser Leu Gl - #n Val Pro Thr Arg Lys                   255          - #       260          - #       265                      - - CGA CCG GCC GAA TCC TCG CCC CAG GTA CCC CC - #C GTT TCC AAC TCG ATG         3202                                                                       Arg Pro Ala Glu Ser Ser Pro Gln Val Pro Pr - #o Val Ser Asn Ser Met               270              - #   275              - #   280                          - - TTG GCC CAC GAT CCG GAC CTC GCT TCC GGC GT - #G CCC GAT TAT GCC TTG         3250                                                                       Leu Ala His Asp Pro Asp Leu Ala Ser Gly Va - #l Pro Asp Tyr Ala Leu           285                 2 - #90                 2 - #95                 3 -      #00                                                                              - - GAC GCC CCG TCC TCG GCC TTT GGC TTC CAT CA - #G GGT AAC CAC CAT        CCG     3298                                                                    Asp Ala Pro Ser Ser Ala Phe Gly Phe His Gl - #n Gly Asn His His Pro                          305  - #               310  - #               315              - - GTC AAT CAT CAC AAC CAC ACC TCC CCC GGG GC - #A CCG TTT GGC TTG GAT         3346                                                                       Val Asn His His Asn His Thr Ser Pro Gly Al - #a Pro Phe Gly Leu Asp                       320      - #           325      - #           330                  - - ACG TTC GGC CTG GGA GAT GAT CCA ATC TTG CC - #C TCC GCG GGC CCC TAC         3394                                                                       Thr Phe Gly Leu Gly Asp Asp Pro Ile Leu Pr - #o Ser Ala Gly Pro Tyr                   335          - #       340          - #       345                      - - CAG TCG CAA TTC ACC TTC TCA CCC AGC GAG TC - #T CCG ATG GCC TCC GGT         3442                                                                       Gln Ser Gln Phe Thr Phe Ser Pro Ser Glu Se - #r Pro Met Ala Ser Gly               350              - #   355              - #   360                          - - CAT CCG TTT GCG AAC CTC TAT TCG CAT ACC CC - #G GTG GCT TCG TCC CTC         3490                                                                       His Pro Phe Ala Asn Leu Tyr Ser His Thr Pr - #o Val Ala Ser Ser Leu           365                 3 - #70                 3 - #75                 3 -      #80                                                                              - - AAC TCG ACG GAT TTC TTC TCT CCA CCG CCA TC - #A GGC TAC CAG TCC        ACG     3538                                                                    Asn Ser Thr Asp Phe Phe Ser Pro Pro Pro Se - #r Gly Tyr Gln Ser Thr                          385  - #               390  - #               395              - - GCA TCC ACG CCG CAG CCC ACC TAC GAC GGG GA - #C CAT TCC GTT TAT TTC         3586                                                                       Ala Ser Thr Pro Gln Pro Thr Tyr Asp Gly As - #p His Ser Val Tyr Phe                       400      - #           405      - #           410                  - - GAT ATG CCG TCG GGC GAC GCG CGC ACC CAG CG - #C CGC ATT CCG AAC TAT         3634                                                                       Asp Met Pro Ser Gly Asp Ala Arg Thr Gln Ar - #g Arg Ile Pro Asn Tyr                   415          - #       420          - #       425                      - - ATT TCG CAT CGG TCC AAC TTG TCT GCT TCG CT - #G CAG CCT CGG TAT ATG         3682                                                                       Ile Ser His Arg Ser Asn Leu Ser Ala Ser Le - #u Gln Pro Arg Tyr Met               430              - #   435              - #   440                          - - TTC AAC CAG AAC AAC CAT GAA CAG GCC AGT TC - #G TCG ACG GTG CAT TCG         3730                                                                       Phe Asn Gln Asn Asn His Glu Gln Ala Ser Se - #r Ser Thr Val His Ser           445                 4 - #50                 4 - #55                 4 -      #60                                                                              - - CCG AGC TAC CCC ATT CCC CAG CCG CAA CAT GT - #G GAC CCC ACT CAG        GTG     3778                                                                    Pro Ser Tyr Pro Ile Pro Gln Pro Gln His Va - #l Asp Pro Thr Gln Val                          465  - #               470  - #               475              - - TTG AAC GCC ACC AAT TAC TCG ACC GGC AAC TC - #C CAC CAT ACC GGC GCC         3826                                                                       Leu Asn Ala Thr Asn Tyr Ser Thr Gly Asn Se - #r His His Thr Gly Ala                       480      - #           485      - #           490                  - - ATG TTT TCA TTT GGA GCC GAT TCA GAT AAC GA - #G GAT GAC GAT GGT CAT         3874                                                                       Met Phe Ser Phe Gly Ala Asp Ser Asp Asn Gl - #u Asp Asp Asp Gly His                   495          - #       500          - #       505                      - - CAG CTG TCC GAG CGG GCT GGT CTG GCG ATG CC - #G ACT GAA TAT GGG GAC         3922                                                                       Gln Leu Ser Glu Arg Ala Gly Leu Ala Met Pr - #o Thr Glu Tyr Gly Asp               510              - #   515              - #   520                          - - GAG GAC GGG TTC TCG TCG GGC ATG CAG TGG GA - #T GGG CAG TTC CCG GGC         3970                                                                       Glu Asp Gly Phe Ser Ser Gly Met Gln Trp As - #p Gly Gln Phe Pro Gly           525                 5 - #30                 5 - #35                 5 -      #40                                                                              - - TCC TTC CAT TCG CTG CCG GGC TTT GGC CCT CA - #A CAT CGC AAG CAT        GTT     4018                                                                    Ser Phe His Ser Leu Pro Gly Phe Gly Pro Gl - #n His Arg Lys His Val                          545  - #               550  - #               555              - - ACC ATC GGG TCC ACG GAC ATG ATG GAC ACC CC - #C GAG GAG TGG AAT CAC         4066                                                                       Thr Ile Gly Ser Thr Asp Met Met Asp Thr Pr - #o Glu Glu Trp Asn His                       560      - #           565      - #           570                  - - GGT GGC AGT TTG GGT CGG ACT CAT GGG TCG GT - #G GCT TCG GTC AGT GAG         4114                                                                       Gly Gly Ser Leu Gly Arg Thr His Gly Ser Va - #l Ala Ser Val Ser Glu                   575          - #       580          - #       585                      - - GTG CGC AAC CGA GAG CAG GAC CCT CGC CGG CA - #G AAG ATT GCC CGC ACC         4162                                                                       Val Arg Asn Arg Glu Gln Asp Pro Arg Arg Gl - #n Lys Ile Ala Arg Thr               590              - #   595              - #   600                          - - ACG TCC ACC CCC AAT ACG GCC CAG CTG TTG CG - #C CAA AGC ATG CAC TCT         4210                                                                       Thr Ser Thr Pro Asn Thr Ala Gln Leu Leu Ar - #g Gln Ser Met His Ser           605                 6 - #10                 6 - #15                 6 -      #20                                                                              - - AAT AAC AAT ACG TCT CAT ACC TCC CCT AAT AC - #G CCG CCC GAG TCC        GCC     4258                                                                    Asn Asn Asn Thr Ser His Thr Ser Pro Asn Th - #r Pro Pro Glu Ser Ala                          625  - #               630  - #               635              - - CTG AGC AGC GCA GTT CCG TCC CGC CCG GCC AG - #T CCC GGG GGC AGC AAG         4306                                                                       Leu Ser Ser Ala Val Pro Ser Arg Pro Ala Se - #r Pro Gly Gly Ser Lys                       640      - #           645      - #           650                  - - AAC GGC GAC CAA GGC AGC AAC GGA CCG ACC AC - #C TGC ACG AAC TGC TTC         4354                                                                       Asn Gly Asp Gln Gly Ser Asn Gly Pro Thr Th - #r Cys Thr Asn Cys Phe                   655          - #       660          - #       665                      - - ACT CAA ACC ACT CCG CTG TGG CGT CGG AAC CC - #A GAG GGC CAG CCA CTG         4402                                                                       Thr Gln Thr Thr Pro Leu Trp Arg Arg Asn Pr - #o Glu Gly Gln Pro Leu               670              - #   675              - #   680                          - - TGC AAT GCC TGC GGG TTG TTT TTG AAA TTG CA - #C GGT GTC GTG CGC CCT         4450                                                                       Cys Asn Ala Cys Gly Leu Phe Leu Lys Leu Hi - #s Gly Val Val Arg Pro           685                 6 - #90                 6 - #95                 7 -      #00                                                                              - - CTG TCC CTG AAA ACG GAC GTT ATC AAA AAG CG - #C AAC CGT AGC AGT        GCC     4498                                                                    Leu Ser Leu Lys Thr Asp Val Ile Lys Lys Ar - #g Asn Arg Ser Ser Ala                          705  - #               710  - #               715              - - AAC AGC TTG GCG GTT GGG ACC TCC CGT GCG TC - #G AAG AAG ACA GCC CGC         4546                                                                       Asn Ser Leu Ala Val Gly Thr Ser Arg Ala Se - #r Lys Lys Thr Ala Arg                       720      - #           725      - #           730                  - - AAG AAC TCG GTG CAG CAA GCA TCC GTC ACG AC - #T CCG ACA TCA AGC CGC         4594                                                                       Lys Asn Ser Val Gln Gln Ala Ser Val Thr Th - #r Pro Thr Ser Ser Arg                   735          - #       740          - #       745                      - - GCT CAG AAT GGG ACT TCC TTC GAA TCC CCG CC - #C GCC GGC TTT AGT GCT         4642                                                                       Ala Gln Asn Gly Thr Ser Phe Glu Ser Pro Pr - #o Ala Gly Phe Ser Ala               750              - #   755              - #   760                          - - GCC GCG GGA CGG TCG AAT GGG GTG GTA CCC AT - #T GCC GCC GCT CCT CCG         4690                                                                       Ala Ala Gly Arg Ser Asn Gly Val Val Pro Il - #e Ala Ala Ala Pro Pro           765                 7 - #70                 7 - #75                 7 -      #80                                                                              - - AAG GCA GCT CCC TCC GCA GCC GCC TCC CCT AG - #C ACG GGC CAG ACC        CGC     4738                                                                    Lys Ala Ala Pro Ser Ala Ala Ala Ser Pro Se - #r Thr Gly Gln Thr Arg                          785  - #               790  - #               795              - - AAC CCG ATC CAG GCT GCC CCG AAA CGT CAA CG - #A CGG CTG GAA AAG GCC         4786                                                                       Asn Pro Ile Gln Ala Ala Pro Lys Arg Gln Ar - #g Arg Leu Glu Lys Ala                       800      - #           805      - #           810                  - - ACG GAG ATG GAA ACG GAC GAG GCT AAC AAG TC - #C GCG GGA GGC CGA TCC         4834                                                                       Thr Glu Met Glu Thr Asp Glu Ala Asn Lys Se - #r Ala Gly Gly Arg Ser                   815          - #       820          - #       825                      - - AAG GTG GTG CCT CTG GCA CCC GCC ATG CCA CC - #G GCA GCA GCC AAT CCG         4882                                                                       Lys Val Val Pro Leu Ala Pro Ala Met Pro Pr - #o Ala Ala Ala Asn Pro               830              - #   835              - #   840                          - - GCG AAC CAT AGT ATT GCC GGA GGC CAA GGG GC - #T AGT CAG GAA TGG GAG         4930                                                                       Ala Asn His Ser Ile Ala Gly Gly Gln Gly Al - #a Ser Gln Glu Trp Glu           845                 8 - #50                 8 - #55                 8 -      #60                                                                              - - TGG TTG ACG ATG AGT CTGTAATGGC CGCGCTTACC TCTCTACTT - #C TCTACACTCG         4985                                                                      Trp Leu Thr Met Ser Leu                                                                       865                                                            - - TTTCTTAATA TCTTTCTTGA ACCCCCCCTT ATATTTTCCC ACCGTTGATG CT -             #ACGCCATG   5045                                                                 - - ACCGATAGAG ATGATGAATA CTGCAACCAA TGGAATCTCG CTAGACGAGA GG -            #TGTTAGAT   5105                                                                 - - GACGTGGCCC GCGATGCACT TAATGAGATA CGAGGAGGTG CAATGCGTTG GT -            #TACGCTAG   5165                                                                 - - TTTAATGGTA ACATGACGAG GGATATTCGC TCTGTTATTT CGGGCTTTGA TC -            #TGTTTCAG   5225                                                                 - - TCTGCGATTT AACAGCGACT GATCCTCTGC TGTGACAATA CACAGCTTGT CT -            #TGTGGTTC   5285                                                                 - - TGTTGTGGCT TTCTGTTTGT TTGGCTGATT TGATTTATGC TTGATACAAT CG -            #CGTCTGTC   5345                                                                 - - CGGACCCCGG CCTTTGTTTT GTTTTCAGTT CTGATTCTTC ACTGTTTCTG AT -            #TCTCTTGT   5405                                                                 - - TCATGTTTTT GATTTGTTCA AGGCTTGGGG CCGGGCAGAA GTGCGCATCT CT -            #GCTTTGTG   5465                                                                 - - TTTTCCGTCA CCGTGCATAG ACGCTGTATG TATATGCTAC AGCAAGATTC TA -            #CTTATCCA   5525                                                                 - - GTCTGAGCCT GTATTCATTG AAGTGTAGCC AGCTGTCGAA TGAGCTTTTT AA -            #CGATATTG   5585                                                                 - - TTTTGTTGAG TAGTCAACAA GTAGTATCTG TATATTCCGG AGTCTAAGTA AG -            #ACACTT     5643                                                                 - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 866 amino - #acids                                                (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                 - -     (ii) MOLECULE TYPE: protein                                           - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - - Met Ser Gly Leu Thr Leu Gly Arg Gly Pro Gl - #y Gly Val Arg Pro        Thr                                                                               1               5 - #                 10 - #                 15             - - Gln Thr Ala Thr Phe Thr Thr His His Pro Se - #r Ala Asp Ala Asp Arg                   20     - #             25     - #             30                  - - Ser Ser Asn Asn Leu Pro Pro Thr Ser Ser Gl - #n Leu Ser Asp Asp Phe               35         - #         40         - #         45                      - - Ser Phe Gly Ser Pro Leu Ser Pro Ala Asp Se - #r Gln Ala His Asp Gly           50             - #     55             - #     60                          - - Leu Leu Gln Asp Ser Leu Phe Pro Glu Trp Gl - #y Ser Gly Ala Pro Arg       65                 - # 70                 - # 75                 - # 80       - - Pro Gly Ile Asp Ser Pro Asp Glu Met Gln Ar - #g Gln Asp Pro Leu Ala                       85 - #                 90 - #                 95              - - Thr Gln Ile Trp Lys Leu Tyr Ser Arg Thr Ly - #s Ala Gln Leu Pro Asn                  100      - #           105      - #           110                  - - Gln Glu Arg Met Glu Asn Leu Thr Trp Arg Me - #t Met Ala Met Ser Leu              115          - #       120          - #       125                      - - Lys Arg Lys Glu Arg Glu Arg Ala Gln Gln Se - #r Met Phe Pro Ala Arg          130              - #   135              - #   140                          - - Arg Gly Ser Ala Gly Pro Ser Gly Ile Ala Gl - #n Leu Arg Ile Ser Asp      145                 1 - #50                 1 - #55                 1 -      #60                                                                              - - Pro Pro Val Ala Thr Gly Asn Pro Gln Ser Th - #r Asp Leu Thr Ala        Asp                                                                                             165  - #               170  - #               175             - - Pro Met Asn Leu Asp Asp Phe Ile Val Pro Ph - #e Glu Ser Pro Ser Asp                  180      - #           185      - #           190                  - - His Pro Ser Pro Ser Ala Val Lys Ile Ser As - #p Ser Thr Ala Ser Ala              195          - #       200          - #       205                      - - Ala Ile Pro Ile Lys Ser Arg Lys Asp Gln Le - #u Arg Asp Ser Thr Pro          210              - #   215              - #   220                          - - Val Pro Ala Ser Phe His His Pro Ala Gln As - #p Gln Arg Lys Asn Ser      225                 2 - #30                 2 - #35                 2 -      #40                                                                              - - Glu Phe Gly Tyr Val Pro Arg Arg Val Arg Ly - #s Thr Ser Ile Asp        Glu                                                                                             245  - #               250  - #               255             - - Arg Gln Phe Phe Ser Leu Gln Val Pro Thr Ar - #g Lys Arg Pro Ala Glu                  260      - #           265      - #           270                  - - Ser Ser Pro Gln Val Pro Pro Val Ser Asn Se - #r Met Leu Ala His Asp              275          - #       280          - #       285                      - - Pro Asp Leu Ala Ser Gly Val Pro Asp Tyr Al - #a Leu Asp Ala Pro Ser          290              - #   295              - #   300                          - - Ser Ala Phe Gly Phe His Gln Gly Asn His Hi - #s Pro Val Asn His His      305                 3 - #10                 3 - #15                 3 -      #20                                                                              - - Asn His Thr Ser Pro Gly Ala Pro Phe Gly Le - #u Asp Thr Phe Gly        Leu                                                                                             325  - #               330  - #               335             - - Gly Asp Asp Pro Ile Leu Pro Ser Ala Gly Pr - #o Tyr Gln Ser Gln Phe                  340      - #           345      - #           350                  - - Thr Phe Ser Pro Ser Glu Ser Pro Met Ala Se - #r Gly His Pro Phe Ala              355          - #       360          - #       365                      - - Asn Leu Tyr Ser His Thr Pro Val Ala Ser Se - #r Leu Asn Ser Thr Asp          370              - #   375              - #   380                          - - Phe Phe Ser Pro Pro Pro Ser Gly Tyr Gln Se - #r Thr Ala Ser Thr Pro      385                 3 - #90                 3 - #95                 4 -      #00                                                                              - - Gln Pro Thr Tyr Asp Gly Asp His Ser Val Ty - #r Phe Asp Met Pro        Ser                                                                                             405  - #               410  - #               415             - - Gly Asp Ala Arg Thr Gln Arg Arg Ile Pro As - #n Tyr Ile Ser His Arg                  420      - #           425      - #           430                  - - Ser Asn Leu Ser Ala Ser Leu Gln Pro Arg Ty - #r Met Phe Asn Gln Asn              435          - #       440          - #       445                      - - Asn His Glu Gln Ala Ser Ser Ser Thr Val Hi - #s Ser Pro Ser Tyr Pro          450              - #   455              - #   460                          - - Ile Pro Gln Pro Gln His Val Asp Pro Thr Gl - #n Val Leu Asn Ala Thr      465                 4 - #70                 4 - #75                 4 -      #80                                                                              - - Asn Tyr Ser Thr Gly Asn Ser His His Thr Gl - #y Ala Met Phe Ser        Phe                                                                                             485  - #               490  - #               495             - - Gly Ala Asp Ser Asp Asn Glu Asp Asp Asp Gl - #y His Gln Leu Ser Glu                  500      - #           505      - #           510                  - - Arg Ala Gly Leu Ala Met Pro Thr Glu Tyr Gl - #y Asp Glu Asp Gly Phe              515          - #       520          - #       525                      - - Ser Ser Gly Met Gln Trp Asp Gly Gln Phe Pr - #o Gly Ser Phe His Ser          530              - #   535              - #   540                          - - Leu Pro Gly Phe Gly Pro Gln His Arg Lys Hi - #s Val Thr Ile Gly Ser      545                 5 - #50                 5 - #55                 5 -      #60                                                                              - - Thr Asp Met Met Asp Thr Pro Glu Glu Trp As - #n His Gly Gly Ser        Leu                                                                                             565  - #               570  - #               575             - - Gly Arg Thr His Gly Ser Val Ala Ser Val Se - #r Glu Val Arg Asn Arg                  580      - #           585      - #           590                  - - Glu Gln Asp Pro Arg Arg Gln Lys Ile Ala Ar - #g Thr Thr Ser Thr Pro              595          - #       600          - #       605                      - - Asn Thr Ala Gln Leu Leu Arg Gln Ser Met Hi - #s Ser Asn Asn Asn Thr          610              - #   615              - #   620                          - - Ser His Thr Ser Pro Asn Thr Pro Pro Glu Se - #r Ala Leu Ser Ser Ala      625                 6 - #30                 6 - #35                 6 -      #40                                                                              - - Val Pro Ser Arg Pro Ala Ser Pro Gly Gly Se - #r Lys Asn Gly Asp        Gln                                                                                             645  - #               650  - #               655             - - Gly Ser Asn Gly Pro Thr Thr Cys Thr Asn Cy - #s Phe Thr Gln Thr Thr                  660      - #           665      - #           670                  - - Pro Leu Trp Arg Arg Asn Pro Glu Gly Gln Pr - #o Leu Cys Asn Ala Cys              675          - #       680          - #       685                      - - Gly Leu Phe Leu Lys Leu His Gly Val Val Ar - #g Pro Leu Ser Leu Lys          690              - #   695              - #   700                          - - Thr Asp Val Ile Lys Lys Arg Asn Arg Ser Se - #r Ala Asn Ser Leu Ala      705                 7 - #10                 7 - #15                 7 -      #20                                                                              - - Val Gly Thr Ser Arg Ala Ser Lys Lys Thr Al - #a Arg Lys Asn Ser        Val                                                                                             725  - #               730  - #               735             - - Gln Gln Ala Ser Val Thr Thr Pro Thr Ser Se - #r Arg Ala Gln Asn Gly                  740      - #           745      - #           750                  - - Thr Ser Phe Glu Ser Pro Pro Ala Gly Phe Se - #r Ala Ala Ala Gly Arg              755          - #       760          - #       765                      - - Ser Asn Gly Val Val Pro Ile Ala Ala Ala Pr - #o Pro Lys Ala Ala Pro          770              - #   775              - #   780                          - - Ser Ala Ala Ala Ser Pro Ser Thr Gly Gln Th - #r Arg Asn Pro Ile Gln      785                 7 - #90                 7 - #95                 8 -      #00                                                                              - - Ala Ala Pro Lys Arg Gln Arg Arg Leu Glu Ly - #s Ala Thr Glu Met        Glu                                                                                             805  - #               810  - #               815             - - Thr Asp Glu Ala Asn Lys Ser Ala Gly Gly Ar - #g Ser Lys Val Val Pro                  820      - #           825      - #           830                  - - Leu Ala Pro Ala Met Pro Pro Ala Ala Ala As - #n Pro Ala Asn His Ser              835          - #       840          - #       845                      - - Ile Ala Gly Gly Gln Gly Ala Ser Gln Glu Tr - #p Glu Trp Leu Thr Met          850              - #   855              - #   860                          - - Ser Leu                                                                  865                                                                          __________________________________________________________________________

We claim:
 1. An Aspergillus oryzae cell comprising a modified areA gene,wherein said modification reduces the synthesis of a functional areAgene product and wherein the modification of the areA gene is(a) adeletion of all or parts of SEQ ID NO:1; or (b) insertion of extra DNAinternally into SEQ ID NO:1.
 2. A method for producing the Aspergillusoryzae cell of claim 1, wherein said method comprises:(a) cloning thearea gene comprising SEQ ID NO:1 from Aspergillus oryzae; (b) producinga DNA construct comprising the area gene wherein an internal part of SEQID NO:1 has been substituted or deleted, or extra DNA has been insertedinto SEQ ID NO:1; (c) transforming said Aspergillus oryzae with theconstruct; and (d) selecting transformants which exhibit a reducedsynthesis of a functional area gene product.
 3. A process for producinga polypeptide comprising the steps of:(a) cultivating the Aspergillusoryzae cell of claim 1; and (b) recovering and purifying thepolypeptide.
 4. The process of claim 3, wherein the Aspergillus oryzaecell has been transformed with a DNA sequence coding for thepolypeptide.
 5. The process of claim 3, wherein the polypeptide is afungal polypeptide.
 6. The process of claim 3, wherein the polypeptideis a secreted protein.
 7. The process of claim 6, wherein thepolypeptide is secreted to the extracellular medium.
 8. An isolated DNAsequence coding for the areA gene from Aspergillus oryzae comprising SEQID NO:1.